There is a specific interaction of human TAFII250 with RAP74, an
essential subunit of the basal transcription factor IIF. Recognition interfaces between TAFII250 and RAP74 have been mapped. In
vivo complementation of a temperature-sensitive TAFII250 cell line reveals that the
RAP74 interaction is critical for cell viability. Because TFIIF is thought to be
responsible for binding and recruiting RNA polymerase II, the ability of TAFII250 to
interact selectively with RAP74 is likely to contribute a critical contact for the
assembly of an active transcription complex (Ruppert, 1995).

Human TFIID TAF (TAFII55) has no known homolog in Drosophila TFIID. TAFII55 has been shown to interact with the largest subunit (TAFII230) of
human TFIID through its central region and with multiple activators (including Sp1,
YY1, USF, CTF, adenoviral E1A, and human immunodeficiency virus-type 1 Tat
proteins) through a distinct amino-terminal domain. The TAFII55-interacting region of
Sp1 is localized to its DNA-binding domain, which is distinct from the glutamine-rich
activation domains previously shown to interact with Drosophila TAFII110. Thus, this
human TFIID TAF may be a co-activator that mediates a response to multiple
activators through a distinct mechanism (Chiang, 1995).

The acetylation of histones increases the accessibility of nucleosomal DNA to transcription factors, relieving transcriptional repression and correlating with the potential for transcriptional activity
in vivo. The characterization of several novel histone acetyltransferases - including the
human GCN5 homolog PCAF (p300/CBP-associated factor), the transcription coactivator
p300/CBP, and TAFII250 - has provided a potential explanation for the relationship between
histone acetylation and transcriptional activation. In addition to histones, however, other components of
the basal transcription machinery might be acetylated by these enzymes and directly affect
transcription. The acetylation of the basal transcriptional machinery for RNA
polymerase II by PCAF, p300 and TAFII250 was examined. All three acetyltransferases can direct the
acetylation of TFIIEbeta and TFIIF; a preferred site of acetylation in TFIIEbeta was identified.
Human TFIIE consists of two subunits, alpha(p56) and beta(p34), which form a heterotetramer (alpha2
beta2) in solution. TFIIE enters the preinitiation complex after RNA
polymerase II and TFIIF, suggesting that TFIIE may interact directly with RNA polymerase II and/or
TFIIF. In addition, TFIIE can facilitate promoter melting either in the presence or absence of
TFIIH and can stimulate TFIIH-dependent phosphorylation of the carboxy-terminal domain of RNA
polymerase II. TFIIF has an essential role in both transcription initiation and
elongation (Imhof, 1997).

Different functional domains of TAFII250 modulate expression of distinct subsets of mammalian genes

The TATA box-binding protein-associated factors (TAFs) are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. In hamster ts13 cells, a single amino acid change in TAF(II)250, which disrupts its acetyl-transferase activity at the restrictive temperature, alters the transcription of specific genes involved in cell cycle control. Likewise, disruption of the amino-terminal kinase domain of TAF(II)250 results in transcriptional defects in ts13 cells. However, it was not known whether the acetyl-transferase or kinase domains of TAF(II)250 modulate specific classes of genes and whether these two domains regulate distinct subsets of genes. High-density gene-profiling has been used to identify mammalian transcripts that require either the TAF(II)250 acetyl-transferase or protein kinase function for proper expression. Transcription of at least 18% of genes are differentially expressed at the restrictive temperature. The promoter region of one of these genes was subsequently characterized, and both upstream elements as well as the core promoter were shown to be TAF(II)250 responsive. Expression of approximately 6% of genes in ts13 cells requires a functional TAF(II)250 amino-terminal kinase domain, but only approximately 1% of these hamster genes also require the TAF(II)250 acetyl-transferase activity. These results suggest that the two TAF(II)250 enzymatic activities are important for regulating largely nonoverlapping sets of genes involved in a wide range of biological functions in vivo (O'Brien, 2000; full text of article).

The TAF N-terminal domain (TAND) of TAF1 includes two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. Previous studies showed that the substitution of yeast TAND1 or TAND2 with the equivalent domain from a Drosophila homologue leads to accumulation of truncated Taf1p in yeast. This study demonstrates that these truncated Taf1p derivatives lack TAND. However, full-length Taf1p and untruncated derivatives are produced in yeast when several Met-to-Ala mutations are introduced in the carboxy-terminus of TAND. In contrast, mutations that reduce expression of full-length TAF1 do not reduce the amount of truncated Taf1p derivatives that are produced. These data suggest that TAND-deficient TAF1 derivatives are produced by initiating translation at alternative initiation sites. In addition, the TAF1 mRNA structure suggests that the TAND-deficient TAF1 derivatives may also be formed in yeast by use of (cryptic) alternative transcription initiation sites. Importantly, TAND-deficient truncated Taf1p appears to be produced at a low level in wild-type yeast as well. Finally, this study also demonstrates that Drosophila TAND2 substitutes functionally for yeast TAND2, but Drosophila TAND1 does not substitute for yeast TAND1 (Kasahara, 2004).

Taf1 interactions with TFIID

Basal transcription factor TFIID comprises the TATA-box-binding protein, TBP, and associated
factors, the TAFIIs. Previous studies have implicated TAFII250 and TAFII150 in core promoter
selectivity of RNA polymerase II. Here, a random DNA binding site selection procedure was used
to identify target sequences for these TAFs. Individually, neither TAFII250 nor TAFII150 singles out a
clearly constrained DNA sequence. However, a TAFII250-TAFII150 complex selects sequences that
match the Initiator (Inr) consensus. When in a trimeric complex with TBP, these TAFs select Inr
sequences at the appropriate distance from the TATA-box. Point mutations that inhibit binding of the
TAFII250-TAFII150 complex also impair Inr function in reconstituted basal transcription reactions,
underscoring the functional relevance of Inr recognition by TAFs. Surprisingly, the precise DNA
sequence at the start site of transcription influences transcriptional regulation by the upstream activator
Sp1. Finally, TAFII150 specifically binds to four-way junction DNA, suggesting that
promoter binding by TFIID may involve recognition of DNA structure as well as primary sequence.
Taken together, these results establish that TAFII250 and TAFII150 bind the Inr directly and that Inr
recognition can determine the responsiveness of a promoter to an activator (Chalkley, 1999).

A picture of TFIID emerges in which its subunit
architecture reflects the organization of basal promoters. In other words, distinct core promoter
elements can be considered to form an array of binding sites for distinct TFIID subunits. Thus, the
TATA-box is bound by TBP, the Inr by a TAFII250-TAFII150 dimer
and the downstream core promoter element (DPE) by a TAFII60-TAFII40 heterotetramer. At present, a role in promoter
recognition for some of the other TAFs cannot be excluded either. Likewise, sequences other than the core motifs uncovered so far, can help determine basal
promoter strength. An interesting feature of the core promoter motifs is their relatively flexible sequence requirements:
many A/T-rich sequences can impart TATA activity; the Inr consensus is rather loose, and partial
DPEs have been described. However, the need for multiple, correctly juxtaposed elements greatly
increases the specificity of TFIID binding. Such combinatorial requirements for binding are not limited
to TFIID but also involve TFIIB, RNA pol II and possibly other basal transcription factors. Thus, a
multitude of individually relatively weak protein-DNA and protein-protein interactions together, make
preinitiation complex formation and initiation of transcription a highly specific process that does not occur randomly on the DNA substrate (Chalkley, 1999 and references).

The main conclusion of the present study, that a TAFII250-TAFII150 complex targets the Inr, agrees
well with results from other studies. (1) It has been demonstrated, by reconstitution of TFIID with
recombinant subunits, that both these TAFs are required for discrimination between Inr-containing and
Inr-less promoters. Promoter selectivity results not only from stabilization
of TFIID-DNA binding, but also from an inhibition of TBP-TATA-box interactions in the absence of a
docking site for TAFII250.
(2) DNA cross-linking and other DNA binding studies have suggested that both these TAFs are in
intimate contact with the promoter DNA, including the Inr region. It should be noted
that the 135 kDa subunit of human TFIID, which can be cross-linked to the AdML promoter is now considered likely to be the human TAFII150. (3) TAFII150 was purified from Drosophila embryos and from human cells as an essential
cofactor for Inr-dependent transcription reconstituted with TAFII150 stripped TFIID. (4) Studies in mammalian and yeast cells have
demonstrated that TAFII250 and its yeast homolog TAFII145, function as core promoter selectivity
factors in vivo (Chalkley, 1999 and references).

The dual requirement for TAFII150 and TAFII250 could be the result of both proteins directly
contacting the Inr. Alternatively, one protein may specifically recognize the Inr sequence, whereas the
other protein stabilizes the complex by making sequence-independent DNA contacts. Since TAFII150
can bind DNA by itself, this protein would be a good candidate for the latter function with TAFII250
providing specific Inr recognition. Finally, it is possible that the binding of TAFII150 and TAFII250 to
one another induces a conformational change that exposes the DNA binding domain. DNA binding by TAFII150 alone does not depend on the precise Inr sequence nor can TAFII150, in
the absence of TAFII250, mediate Inr function. Since the DNase I footprint of TAFII150 on the AdML or the Drosophila
hsp70 promoter extends significantly beyond the Inr element, additional DNA sequences appear to
contribute to TAFII150 binding. However,
a critical consensus sequence for TAFII150 DNA binding could not be determined. Instead, the results indicate that DNA
secondary structure can be an important determinant for TAFII150 binding. Association with TAFII250 inhibits binding of TAFII150 to 4WJ DNA. Interestingly, TAFII250 has a
similar inhibitory effect on DNA binding by TBP). It is probable that docking of TAFII250
on a correctly positioned Inr element neutralizes its inhibition of DNA binding by associated proteins
TBP and TAFII150. Indeed, efficient DNA binding of
recombinant TAFII250-TAFII150, TBP-TAFII250 and TBP-TAFII250-TAFII150 complexes to
Inr-containing promoters has been demonstrated (Chalkley, 1999 and references).

The functional significance of the recognition of structured DNA by TAFII150 is unclear at this
moment. It is possible that the Py-rich Inr region adopts a secondary structure that deviates from
regular B-form DNA. It has also been proposed that the promoter DNA wraps around the TFIID
complex and forms a nucleosome-like structure. DNA wrapping around
TFIID may create a DNA crossover point that is recognized by TAFII150, which may stabilize a
stereo-specific nucleoprotein structure (Chalkley, 1999 and references).

Gene-specific activators are the main regulators of gene expression in eukaryotic cells. This has led to
the perception that transcription is controlled strictly via enhancers and that core promoters are merely
passive docking sites for the basal machinery. However, a number of recent reports, including this
study, emphasize that the basal promoter structure can be a major determinant of the effects elicited by
a transcriptional activator. When fused to the GAL4 DNA binding domain, the activation domain of
VP16 or one of the SP1 activation domains, activator constructs show different activation profiles on distinct core
promoters. In Drosophila embryos it has been elegantly demonstrated that
the core promoter structure can determine selectivity for particular natural activators in vivo. Finally, the results presented here show that the precise sequence at the start site of
transcription can significantly influence the level of activation achieved by the transcriptional activator
Sp1 (Chalkley, 1999 and references).

Taken together, these studies demonstrate that recognition of the basal promoter can play a prominent
role during transcriptional activation by upstream binding regulators. Thus, the great diversity among
natural core promoters might allow different genes to respond differently to a particular activator.
How can the core promoter sequence influence the responsiveness to activating signals? One
attractive possibility is that TFIID adopts distinct conformations on different core promoters that are
either more or less receptive to activating signals from particular activators. Such a mechanism is not
unprecedented since a number of examples of DNA-induced allosteric effects during transcriptional
regulation have been described. Reversibly, activators can induce extended TFIID-DNA
contacts, probably by changes in the TFIID conformation. It will be
important to obtain direct proof of isomerization of TFIID by techniques such as atomic force
microscopy or protease sensitivity mapping. In summary, the Inr is specifically recognized by a TAFII250-TAFII150
complex. Surprisingly, the Inr sequence not only determines the basal promoter strength but also
influences the responsiveness of a promoter to activating signals. These results indicate that recognition
of the core promoter may be more intimately tied to the regulation of transcription by activators than
previously anticipated (Chalkley, 1999 and references).

Recombinant hTAFII250 binds directly to TBP
both in vitro and in yeast, and participates in the formation of the TFIID complex. This largest TAF may therefore play a central role in TFIID assembly by interacting with both TBP and other TAFs, as well as serving to link the control of transcription to the cell cycle (Ruppert, 1993).

Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex. Phosphorylation of yeast TFIIA stimulates TFIIA.TBP.TATA complex formation and transcription activation in vivo. Human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits. Alanine substitution mutation of serine residues 316 and 321 in TFIIA alphabeta reduces TFIIA phosphorylation significantly in vivo. Additional alanine substitutions at serines 280 and 281 reduces phosphorylation to undetectable levels. Mutation of all four serine residues reduces the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function. In vitro, holo-TFIID and TBP-associated factor 250 (TAF(II)250) phosphorylates TFIIA on the beta subunit. Mutation of the four serines required for in vivo phosphorylation eliminates TFIID and TAF(II)250 phosphorylation in vitro. The NH(2)-terminal kinase domain of TAF(II)250 is sufficient for TFIIA phosphorylation, and this activity is inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAF(II)250 interaction or tumor suppressor activity. TFIIA phosphorylation has little effect on the TFIIA.TBP.TATA complex in electrophoretic mobility shift assay. However, phosphorylation of TFIIA containing a gamma subunit Y65A mutation strongly stimulates TFIIA.TBP.TATA complex formation. TFIIA-gammaY65A is defective for binding to the beta-sheet domain of TBP identified in the crystal structure. These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA.TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure (Solow, 2001).

TFIID, a multiprotein complex composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), can directly recognize core promoter elements and mediate transcriptional activation. The TAF N-terminal domain (TAND) of TAF1 may play a significant role in these two principal TFIID functions by regulating the access of TBP to the TATA element. In yeast, TAND consists of two subdomains, TAND1 (10-37 amino acids (aa)) and TAND2 (46-71 aa), which interact with the concave and convex surfaces of TBP, respectively. Another region located on the C-terminal side of TAND2 (82-139 aa) can also bind to TBP and induce transcriptional activation when tethered to DNA as a GAL4 fusion protein. As these properties are the same as those of TAND1, this region has been denoted as TAND3. Detailed mutational analyses revealed that three blocks of hydrophobic amino acid residues located within TAND3 are required not only for TBP binding and transcriptional activation but also for supporting cell growth and the efficient transcription of a subset of genes. The surface of TBP recognized by TAND3 is broader than that recognized by TAND1, although these regions overlap prtially. Supporting these observations is that TAND1 can be at least partly functionally substituted by TAND3 (Takahata, 2003).

General transcription factor TFIID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription. The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes. The Drosophila TAND region of TAF1 (residues 11-77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP. In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro. Structural and functional studies have been performed of yeast TAND1 and TAND2 regions (residues 10-37, and 46-71, respectively). NMR data show that, in yeast, TAND1 contains two alpha-helices (residues 16-23, 30-36) and TAND2 forms a mini beta-sheet structure (residues 53-56, 61-64). These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively. Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex alpha-helix 2, overlaps with the mini beta-sheet region (Mal, 2004).

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. This study used RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, it was found that TAF4 rather than TBP or TAF1 plays the most critical role in maintaining stability of the complex. This analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on these results, it is proposed that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID (Wright, 2006; Full text of article).

Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase II-transcribed genes. The downstream core element (DCE) was discovered in the human beta-globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). The DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF(II)250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex (Lee, 2004).

Interaction of TFIID with histones

Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, it has been shown that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacts with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone is essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persists on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions (Kanno, 2004).

Given that H3 is a preferred substrate for many HATs in in vitro assays, the absence of FRET between H3 and Brd2 was striking. This could not be explained by a lack of H3 acetylation, since HeLa cells contained significant amounts of acetylated H3. This led to a test of another bromodomain protein, TAFII250, for interaction with H3. TAFII250 is a component of the basal transcription factor TFIID, and its double bromodomain module binds to an acetylated H4 peptide in vitro and acetylated H3 in a reconstituted chromatin target (Agalioti, 2002). Transfected CFP-TAFII250 associated with other components of TFIID in HeLa cells, indicating that it was incorporated into a stable TFIID complex. It was found that TAFII250 produced a significant FRET signal with H3 as well as H4 and to a lesser extent H2B but not with H2A. Because TAFII250 has a HAT activity and contains a histone recognition site outside the bromodomains (Mizzen, 1996), deletions were tested that removed the HAT region, but retained the bromodomains. The deletions produced FRET with H3 and H4 as well as did full length TAFII250. It is of note that these deletions also lacked the TBP binding surface, and TAFII250-BD is further devoid of the putative HMG-like region, indicating that DNA binding of TAFII250 is dispensable for its histone association. Thus, H3 is recognized by the bromodomains of TAFII250 but not of Brd2. Moreover, in contrast to FRET with Brd2, both mutants H4-K(5,12)G and H4-K(8,16)G partially reduced FRET with TAFII250, indicating that K5/K12 and K8/K16 of H4 both critically contribute to the interaction with TAFII250. Peptide precipitation analysis showed that TAFII250 bound H4 peptides mono-acetylated at K8, K12, or K16 as well as H3 peptides acetylated at K14. It is concluded that specific lysine residues of both H3 and H4 are recognized by the TAFII250 bromodomains in vivo and that TAFII250 has broader recognition specificity than Brd2 (Kanno, 2004).

Plant growth and development are sensitive to light. Light-responsive DNA-binding transcription factors have been functionally identified. However, how transcription initiation complex integrates light signals from enhancer-bound transcription factors remains unknown. This work characterizes mutations within the Arabidopsis HAF2 gene encoding TATA-binding protein-associated factor TAF1 (or TAF(II)250). The mutation of HAF2 induced decreases on chlorophyll accumulation, light-induced mRNA levels, and promoter activity. Genetic analysis indicated that HAF2 is involved in the pathways of both red/far-red and blue light signals. Double mutants between haf2-1 and hy5-1, a mutation of a light signaling positive DNA-binding transcription factor gene, had a synergistic effect on photomorphogenic traits and light-activated gene expression under different light wavelengths, suggesting that HAF2 is required for interaction with additional light-responsive DNA-binding transcription factors to fully respond to light induction. Chromatin immunoprecipitation assays showed that the mutation of HAF2 reduced acetylation of histone H3 in light-responsive promoters. In addition, transcriptome analysis showed that the mutation altered the expression of about 9% of genes in young leaves. These data indicate that TAF1 encoded by the Arabidopsis HAF2 gene functions as a coactivator capable of integrating light signals and acetylating histones to activate light-induced gene transcription (Bertrand, 2005).

TAFII250 and cell cycle regulation

CCG1/TAFII250, the largest subunit of the TFIID complex, is mutated in specific temperature sensitive (ts) cell cycle
mutants that have a promoter-selective
transcriptional defect. A series of deletion mutants of TAFII250 were prepared
and transfected into the cellular mutants, in order to identify functional domains of TAFII250
required for the complementation of ts mutation. The
minimum size of TAFII250, essential for complementing the ts mutation,
possesses one proline cluster, an HMG1-like domain, and a nuclear localization signal,
but lacks the bromo domains and the acidic phosphorylation sites for casein
kinase II common to transcriptional activators. It encodes a protein of 140 kDa. The minimal protein complementing the ts muation corresponds to yeast TAFII145, the yeast homolog of
human TAFII250. Truncated TAFII250 binds to TBP, creating its own TFIID complex different
from that of the endogenous mutated TAFII250 in ts+ transformants of tsBN462 cells (Noguchi, 1994).

Stimulation of Sp1-mediated transcription
by RB (Drosophila homolog: Retinoblastoma-family protein)is partially abrogated at the nonpermissive temperature in cultured cells. These
cells contain a temperature-sensitive mutation in the TATA-binding protein-associated
factor TAFII250, first identified as the cell cycle regulatory protein CCG1. The
stimulation of Sp1-mediated transcription by RB in mutant hamster cells at the nonpermissive
temperature can be restored by the introduction of wild-type human TAFII250.
Furthermore, RB binds directly to hTAFII250 in vitro and in vivo.
These results suggest that RB can confer transcriptional regulation and possibly cell
cycle control and tumor suppression through an interaction with TFIID, in particular
with TAFII250 (Shao, 1995).

The retinoblastoma tumor suppressor protein, Rb, interacts directly with the largest TATA-binding
protein-associated factor, TAFII250, through multiple regions in each protein. To define the potential role(s)
of this interaction, it was necessary to examine whether Rb could regulate the intrinsic, bipartite kinase activity of TAFII250. Rb is able to inhibit the kinase activity of immunopurified and gel-purified recombinant
TAFII250. Rb inhibits the autophosphorylation of TAFII250 as well as its phosphorylation of the RAP74
subunit of TFIIF in a dose-responsive manner. Inhibition of TAFII250 kinase activity involves the Rb pocket
(amino acids 379 to 928) but not its amino terminus. In addition, Rb appears to specifically inhibit the
amino-terminal kinase domain of TAFII250 through a direct protein-protein interaction. Two different tumor-derived Rb pocket mutants, C706F and Deltaex22, are functionally
defective for kinase inhibition, even though they are able to bind the amino terminus of TAFII250. These results
suggest a novel mechanism of transcriptional regulation by Rb, involving direct interaction with TAFII250
and inhibition of its ability to phosphorylate itself, RAP74, and possibly other targets (Siegert, 1999).

The TAFII250 subunit of the human transcription factor IID (TFIID) rescues the
temperature-sensitive hamster cell line and overcomes a G1 arrest. Investigation
of the transcriptional properties of nuclear extracts of the mutant cell line in shows that activation
by the site-specific regulators Sp1 and Gal4VP16 is temperature sensitive in
extracts, whereas basal transcription remains unaffected. This transcriptional defect
can be rescued by purified human TFIID or by expression of wild-type TAFII250 in
mutant cells. Expression from the cyclin A (see Drosophila Cyclin A) is temperature
sensitive in these mutant cells. Thus, the mutation in TAFII250 appears to have
gene-specific effects that may lead to the mutant cell cycle phenotype (Wang, 1994).

A specific mutation in TAFII250, the largest subunit of the transcription factor TFIID, disrupts cell
growth control in the temperature-sensitive mutant hamster cell line ts13. Transcription from the cyclin
A and D1(but not the c-fos and myc promoters) is also dramatically reduced in ts13 cells at the
nonpermissive temperature. These findings provide an intriguing link between TAF-mediated
transcriptional regulation and cell cycle progression. An enhancer
element in the cyclin A promoter (TSRE) has been mapped that responds to mutations in TAFII250. An analysis of
chimeric promoter constructs reveals that the cyclin A TSRE can confer TAFII250 dependence to the
core promoter of c-fos. Reciprocal hybrid promoter constructs suggest that TAFII250 also
contributes to the transcriptional properties of the cyclin A core promoter. The cellular activators that specifically bind to the TSRE and mediate transcription in a
TAFII250-dependent manner have been purified and
identified. TSRE-binding proteins
include members of the activating transcription factor (ATF) family. These results suggest that the ts13
mutation of TAFII250 has compromised the ability of TFIID to mediate activation of transcription by
specific enhancer factors such as ATF, as well as its ability to perform certain core promoter functions. These
defects in TAFII250 apparently result in the down-regulation of key molecules, such as cyclin A, which
may be responsible for the ts13 cell cycle arrest phenotype (Wang, 1997).

The largest subunit of the human transcription factor TFIID, TAFII250,
contains serine/threonine kinase domains that can autophosphorylate and transphosphorylate the large
subunit of the basal factor TFIIF. Here, the regions of the N-terminal kinase domain
(amino acids 1-414) necessary for kinase activity are identified and its function in vivo is examined. Point mutations
within two patches of amino acids in the kinase domain decrease both autophosphorylation and
transphosphorylation activities. TAFII250-bearing mutations within the
N-terminal kinase domain exhibit a significantly reduced ability to rescue ts13 cells that express a
temperature-sensitive TAFII250. Moreover, transcription from the cyclin A and cdc2 promoters
becomes impaired when cotransfected with hTAFII250 containing inactive forms of the N-terminal
kinase domain. These results suggest that the TAFII250 kinase activity is required to direct transcription
of at least some genes in vivo (O'Brien, 1998).

MDM2 proto-oncogene expression is aberrant in many human tumors. Its normal role is to modulate
the functions of p53. The N terminus of MDM2 interacts with p53, whereas the properties of the rest
of the molecule are poorly understood. MDM2 is shown to bind to the general transcription factor
TFIID in vivo. The C-terminal Ring finger interacts with TAFII250/CCG1, and the central acidic
domain interacts with TBP. Expression of MDM2 activates the cyclin A gene promoter but not c-fos,
showing that the effects of MDM2 are specific. Deletion of the C-terminal region of MDM2 abolishes
activation, showing that the C-terminal domain of MDM2 is functionally important. Increasing MDM2 expression to higher levels inhibits the cyclin A promoter. Inhibition appears to result
from titration of general transcription factors because MDM2 overexpression inhibits c-fos as well as
other promoters in vivo and basal transcription in vitro. The mechanisms of repression of the cyclin A
and fos promoters appear to be different. Cyclin A repression is lost by deleting the C terminus,
whereas that of c-fos is lost by removal of the acidic domain. These results reinforce the conclusion
that the C terminus of MDM2 mediates effects on the cyclin A promoter. MDM2 transformed cells
contain elevated levels of cyclin A mRNA, showing that activation occurs under physiological
conditions. There is a positive correlation between MDM2 binding to TAFII250 and MDM2 activation
of the cyclin A promoter. The C-terminal region of MDM2, which contains the Ring finger, interacts
with TAFII250 and is required for regulation of the cyclin A promoter by MDM2. These results link the
activity of MDM2, a transforming protein implicated in many human tumors, with cyclin A, a regulator
of the cell cycle (Leveillard, 1997).

The TATA-binding protein (TBP)-associated factor TAF(II)250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAF(II)250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G(1). At the nonpermissive temperature (39.5°C), transcription from only a subset of protein encoding genes, including the G(1) cyclins, is dramatically reduced in the mutant cells. This study demonstrates that the ability of the ts13 allele of TAF(II)250 to acetylate histones in vitro is temperature sensitive suggesting that this enzymatic activity is compromised at 39.5°C in the mutant cells. Mutagenesis of a putative acetyl coenzyme A binding site produced a TAF(II)250 protein that displayed significantly reduced histone acetyltransferase activity but retained TBP and TAF(II)150 binding. Expression of this mutant in ts13 cells was unable to complement the cell cycle arrest or transcriptional defect observed at 39.5°C. These data suggest that TAF(II)250 acetyltransferase activity is required for cell cycle progression and regulates the expression of essential proliferative control genes (Dunphy, 2000).

Doxorubicin (DOX) is a DNA topoisomerase II inhibitor widely used in anticancer treatment, however, it can lead to irreversible cardiac damage with severe debilitation. TBP-binding associated factor 1 (TAF1) is increased in DOX damaged hearts in vivo and in cardiomyocytes in vitro. To identify the functional role for TAF1 in DOX-treated heart wild type and mutant TAF1 was overexpressed in H9c2 cells. Overexpression of wild-type TAF1, but not N-terminal kinase domain mutants, increased tolerance to DOX in confluent cells. DOX treatment can cause prolonged G1 arrest. Increased cdk2 activity coupled to increased cyclin E protein and decreased p21(waf1Cip1) and p27(Kip1) protein was found to correlate only with increased DOX tolerance and wild-type TAF1. DOX sensitivity was restored when the cdk2-inhibitor Roscovitine was co-administered with DOX. Overexpression of cdk2-alone increased resistance to DOX. Thus, TAF1 induced DOX tolerance in confluent cells through an increase in cdk2 activity is directed by the TAF1 N-terminal domain. These studies suggest new avenues for myocardial protection against DOX toxicity and suggest a role for cdk2 in chemorefractory cells (Servent, 2004)

Phosphorylation on Thr-55 by TAF1 mediates degradation of p53: a role for TAF1 in cell G1 progression

The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, the link between TAF1 and p53 was investigated. TAF1 was found to induce G1 progression in a p53-dependent manner. TAF1 interacts with and phosphorylates p53 at Thr-55 in vivo. Substitution of Thr-55 with an alanine residue (T55A) stabilizes p53 and impairs the ability of TAF1 to induce G1 progression. Furthermore, both RNAi-mediated TAF1 ablation and apigenin-mediated inhibition of the kinase activity of TAF1 markedly reduces Thr-55 phosphorylation. Thus, phosphorylation and the resultant degradation of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation is reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage (Li, 2004).

TAFII250 interaction with Tat

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes.
Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has been
unknown. Repression results from the interaction of Tat with the TAFII250 component of the general
transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl
transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat,
including the MHC class I promoter, are dependent on TAFII250, whereas those that are not repressed by Tat, such as
SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription
could be one mechanism by which HIV avoids immune surveillance (Weissman, 1998).

TAF(II)250-independent transcription can be conferred on a TAF(II)250-dependent basal promoter by upstream activators

TAF(II)250, a component of the general transcription factor, TFIID, is required for the transcription of a subset of genes, including those involved in regulating cell cycle progression. The tsBN462 cell line, with a temperature-sensitive mutation of TAF(II)250, grows normally at 32°C, but when grown at 39.5°C, it differentially arrests transcription of many, but not all, genes. The present studies examine the basis for the requirement for TAF(II)250. The basal promoter of a major histocompatibility complex class I gene requires TAF(II)250. This dependence can be overcome by select upstream regulatory elements but not by basal promoter elements. Thus, the coactivator CIITA rescues the basal promoter from the requirement for TAF(II)250, whereas introduction of a canonical TATAA box does not. Similarly, the SV40 basal promoter is shown to require TAF(II)250, and the presence of the 72-base pair enhancer overcomes this requirement. Furthermore, the SV40 72-base pair enhancer when placed upstream of the basal class I promoter renders it independent of TAF(II)250. These data suggest that the assembly of transcription initiation complexes is dynamic and can be modulated by specific transcription factors (Weissman, 2000).

TAFII55 binding to TAFII250 inhibits its acetyltransferase activity

The general transcription factor, TFIID, consists of the TATA-binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. One of the TAFs, TAF(II)250, has acetyltransferase (AT) activity that is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAF(II)250, a yeast two-hybrid library was screened with a TAF(II)250 segment (amino acids 848-1279) that spanned part of its AT domain and it's the domain that binds to the protein, RAP74. The TFIID component, TAF(II)55, was isolated and found to interact predominantly with the RAP74-binding domain. TAF(II)55 binding to TAF(II)250 inhibits its AT activity. Importantly, the addition of recombinant TAF(II)55 to in vitro transcription assays inhibits TAF(II)250-dependent MHC class I transcription. Thus, TAF(II)55 is capable of regulating TAF(II)250 function by modulating its AT activity (Gegonne, 2001).

Transactivation mediated by B-Myb is dependent on TAF(II)250

B-Myb is a highly conserved member of the Myb family of transcription factors, which has been implicated in cell cycle regulation. B-Myb is expressed in most proliferating cells and its activity is highly regulated around the G1/S-phase border of the cell cycle. It is generally assumed that B-Myb regulates the expression of genes that are crucial for cell proliferation; however, the identity of these genes, the molecular mechanisms by which B-Myb stimulates their expression and the involvement of other proteins have not been sufficiently clarified. The hamster cell line ts13 was used as a tool to demonstrate a functional link between B-Myb and the coactivator TAF(II)250, a key component of the transcriptional machinery which itself is essential for cell proliferation. ts13 cells express a point-mutated version of TAF(II)250 whose intrinsic histone acetyl transferase activity is temperature sensitive. Transactivation of Myb-responsive reporter genes by B-Myb is temperature-dependent in ts13 cells but not in ts13 cells, which have been rescued by transfection with an expression vector for wild-type TAF(II)250. Furthermore, B-Myb and TAF(II)250 can be coprecipitated, suggesting that both proteins are present in a complex. The formation of this complex is dependent on the DNA-binding domain of B-Myb and not on its transactivation domain. Taken together, these observations provide the first evidence that the coactivator TAF(II)250 is involved in the activation of Myb responsive promoters by B-Myb. The finding that B-Myb transactivation is dependent on a key coactivator involved in cell cycle control is consistent with and strengthens the idea that B-Myb plays a crucial role as a transcription factor in proliferating cells (Bartusel, 2003).

The basic leucine zipper domain of c-Jun functions in transcriptional activation through interaction with the N terminus of human TAF(II)250

c-Jun binds directly to the N-terminal 163 amino acids of Homo sapiens TATA-binding protein-associated factor-1 (hsTAF1), causing a derepression of transcription factor IID (TFIID)-driven transcription. This region of hsTAF1 binds TATA-binding protein to repress TFIID DNA binding and transcription. The basic leucine zipper domain of c-Jun, which allows for DNA binding and homodimerization, is necessary and sufficient for interaction with hsTAF1. Interestingly, the isolated basic leucine zipper domain of c-Jun is able to derepress TFIID-directed basal transcription in vitro. Moreover, when the N-terminal region of hsTAF1 is added to in vitro transcription reactions and overexpressed in cells, it blocks c-Jun activation. c-Fos, another basic leucine zipper protein, does not interact with hsTAF1, but c-Fos/c-Jun heterodimers bind the N terminus of hsTAF1. These studies show that, in addition to dimerization and DNA binding, the well characterized basic leucine zipper domain of c-Jun functions in transcriptional activation by binding to the N terminus of hsTAF1 to derepress transcription (Lively, 2004).

Taf1 and the transcriptional regulation of Cyclin D

Sp1-mediated transcription is stimulated by Rb and repressed by cyclin D1. The stimulation of Sp1 transcriptional activity by Rb is conferred, in part, through a direct interaction with the TBP-associated factor TAF(II)250. This study investigated the mechanism(s) through which cyclin D1 represses Sp1. The ability of cyclin D1 to regulate transcription mediated by Gal4-Sp1 fusion proteins, which contain the Gal4 DNA-binding domain and Sp1 trans-activation domain(s), was examined. The domain of Sp1 sufficient to confer repression by cyclin D1 was mapped to a region important for interaction with TAF(II)110. TAF(II)250-cyclin D1 complexes can be immunoprecipitated from mammalian and baculovirus-infected insect cells and, recombinant GST-TAF(II)250 (amino acids 1-434) associates with cyclin D1 in vitro. Moreover, the overexpression of Rb or CDK4 reduces the level of TAF(II)250-cyclin D1 complex. The amino terminus of cyclin D1 (amino acids 1-100) is sufficient for association with TAF(II)250 and for repressing Sp1-mediated transcription. Taken together, the results suggest that cyclin D1 may regulate transcription by interacting directly or indirectly with TAF(II)250 (Adnane, 1999).

A missense mutation within the histone acetyltransferase (HAT) domain of the TATA binding protein-associated factor TAF1 induces ts13 cells to undergo a late G(1) arrest and decreases cyclin D1 transcription. TAF1 mutants (Delta844-850 and Delta848-850, from which amino acids 844 through 850 and 848 through 850 have been deleted, respectively) deficient in HAT activity are unable to complement the ts13 defect in cell proliferation and cyclin D1 transcription. Chromatin immunoprecipitation assays revealed that histone H3 acetylation is reduced at the cyclin D1 promoter but not the c-fos promoter upon inactivation of TAF1 in ts13 cells. The hypoacetylation of H3 at the cyclin D1 promoter is reversed by treatment with trichostatin A (TSA), a histone deacetylase inhibitor, or by expression of TAF1 proteins that retain HAT activity. Transcription of a chimeric promoter containing the Sp1 sites of cyclin D1 and c-fos core remain TAF1 dependent in ts13 cells. Treatment with TSA restores full activity to the cyclin D1-c-fos chimera at 39.5 degrees C. In vivo genomic footprinting experiments indicate that protein-DNA interactions at the Sp1 sites of the cyclin D1 promoter are compromised at 39.5 degrees C in ts13 cells. These data have led to a hypothesis that TAF1-dependent histone acetylation facilitates transcription factor binding to the Sp1 sites, thereby activating cyclin D1 transcription and ultimately G(1)-to-S-phase progression (Hilton, 2005).

Cyclin D1 is an oncogene that regulates progression through the G(1) phase of the cell cycle. A temperature-sensitive missense mutation in the transcription factor TAF1/TAF(II)250 induces the mutant ts13 cells to arrest in late G(1) by decreasing transcription of cell cycle regulators, including cyclin D1. Evidence is provided that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, protein-DNA interactions have been identified within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which has been termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 degrees C but not at 37 degrees C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. The finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter (Hilton, 2003).

Taf1 and checkpoint response

Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. A cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest can be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM are able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response (Buchmann, 2004).

Transcription consists of a series of highly regulated steps: assembly of a preinitiation complex (PIC) at the promoter nucleated by TFIID, followed by initiation, elongation, and termination. The present study has focused on the role of the TFIID component, TAF7, in regulating transcription initiation. In TFIID, TAF7 binds to TAF1 and inhibits its intrinsic acetyl transferase activity. It is now reported that although TAF7 remains bound to TAF1 and associated with TFIID during the formation of the PIC, TAF7 dissociates from the PIC upon transcription initiation. Entry of polymerase II into the assembling PIC is associated with TAF1 and TAF7 phosphorylation, coincident with TAF7 release. It is proposed that the TFIID composition is dynamic and that TAF7 functions as a check-point regulator suppressing premature transcription initiation until PIC assembly is complete (Gegonne, 2006).

Functional substitution for TAF(II)250 by a retroposed homolog that is expressed in human spermatogenesis

TAF(II)250 is expressed from the human X chromosome, at least in somatic cells. In male meiosis, however, the sex chromosomes are transcriptionally silenced, while the autosomes remain active. How then are protein-encoding genes transcribed during human male meiosis? A novel autosomal human gene, TAF1L, is homologous to TAF(II)250 and is expressed specifically in the testis, apparently in germ cells. It is hypothesized that during male meiosis, transcription of protein-encoding genes relies upon TAF1L as a functional substitute for TAF(II)250. Like TAF(II)250, the human TAF1L protein can bind directly to TATA-binding protein, an essential component of TFIID. Most importantly, transfection with human TAF1L rescued the temperature-sensitive lethality of a hamster cell line mutant in TAF(II)250. TAF1L lacks introns and evidently arose by retroposition of a processed TAF(II)250 mRNA during primate evolution. The observation that TAF1L can functionally replace TAF(II)250 provides experimental support for the hypothesis that during male meiosis, autosomes provide cellular functions usually supplied by the X chromosome in somatic cells (Wang. 2002).

Regulated expression of TAF1 in 1-cell mouse embryos

TATA binding protein (TBP) associated factor 1 (TAF1) is a member of the general transcription machinery. Interference in the function of TAF1 causes a broad transcriptional defect in early development. To explore possible roles of TAF1 in embryonic transcriptional silence and zygotic genome activation, the expression of TAF1 was examined in 1-cell mouse embryos. Using an immunofluorescence assay, TAF1 was not detected in embryos in the first few hours after fertilization. TAF1 appeared in pronuclei 6 h post-fertilization and reached a relatively high level before zygotic genome activation. These data show that besides TBP, another critical member of the general transcription machinery such as TAF1 is also absent or at an extremely low level at the outset of development. Combined deficiency in critical members of the general transcription machinery may account for embryonic transcriptional silence (Wang, 2006).

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation, or apoptosis in numerous cancer cell types both in vivo and in vitro. These dramatic effects are the result of a specific reprogramming of gene expression. However, the mechanism by which these agents activate the transcription of some genes, such as p21(WAF1), but repress others, such as cyclin D1, is currently unknown. The human SRC gene has been studied as a model for HDI-mediated transcriptional repression. Both the tissue-specific and housekeeping SRC promoters were equally repressed by HDIs. Despite an overt dissimilarity, both SRC promoters do share similar core promoter elements and transcription is TAF1 dependent. Detailed analysis of the SRC promoters suggested that both core and proximal promoter elements are responsible for HDI-mediated repression. This was confirmed in a series of promoter-swapping experiments with the HDI-inducible, TAF1-independent p21(WAF1) promoter. Remarkably, all the SRC-p21(WAF1) chimeric promoter constructs are not only repressed by HDIs but also dependent on TAF1. Together these experiments suggest that the overall promoter architecture, rather than discrete response elements, is responsible for HDI-mediated repression, and they implicate core promoter elements in particular as potential mediators of this response (Dehm, 2004).

Reduced neuron-specific expression of the TAF1 gene is associated with X-linked dystonia-parkinsonism

X-linked dystonia-parkinsonism (XDP) is a movement disorder endemic to the Philippines. The disease locus, DYT3, has been mapped to Xq13.1. In a search for the causative gene, genomic sequencing analysis, followed by expression analysis of XDP brain tissues, was performed. A disease-specific SVA (short interspersed nuclear element, variable number of tandem repeats, and Alu composite) retrotransposon insertion was found in an intron of the TATA-binding protein-associated factor 1 gene (TAF1), which encodes the largest component of the TFIID complex, and significantly decreased expression levels of TAF1 and the dopamine receptor D2 gene (DRD2) in the caudate nucleus. An abnormal pattern of DNA methylation was identified in the retrotransposon in the genome from the patient's caudate, which could account for decreased expression of TAF1. These findings suggest that the reduced neuron-specific expression of the TAF1 gene is associated with XDP (Makino, 2007).